Analytical Separations

1
Analytical Separations

• Chromatographic Principle

2
Chromatographic Separations

• Column Chromatography
• -the stationary phase is held in a narrow
  tube.
• -the mobile phase is forced through the tube
  
• under pressure or by gravity.
• Planar Chromatography
• - the stationary phase is supported on a flat
  plate
• or in the pores of a paper
• - the mobile phase moves through the
  stationary
• phase by capillary action or by gravity.

3
Types of Chromatography

• Adsorption Chromatography
• Partition Chromatography
• Ion Exchange Chromatography
• Molecular Exclusion Chromatography
• Affinity Chromatography

4
Adsorption Chromatography

• One of the oldest types of chromatography around.
  
• It utilizes a mobile liquid or gaseous phase that
  is adsorbed onto the surface of a stationary
  solid phase.
• The equilibrium between the mobile and stationary
  phase accounts for the separation of different
  solutes.

5
Partition Chromatography

• Based on a thin film formed on the surface of a
  solid support by a liquid stationary phase.
• Solute equilibriates between the mobile phase and
  the stationary liquid.

6
Ion Exchange Chromatography

• The use of a resin (the stationary phase) is used
  to covalently attach anions or cations onto it.
• Solute ions of the opposite charge in the mobile
  liquid phase are attracted to the resin by
  electrostatic forces.

7
Molecular Exclusion Chromatography

• Lacks an attractive interaction between the
  stationary phase and solute.
• The mobile phase passes through a porous gel
  which separates the molecules according to its
  size.
• The pores are normally small.
• The larger molecules pass through the column at a
  faster rate than the smaller ones.

8
Separating Ions by Ion Exchange

• Ion exchange resin
• -high molecular weight polymers
• -large numbers of an ionic functional group
• -cation exchange resin
• -anion exchange resin

9
Ion Exchange Equilibria
K?strong tendency for binding Ca2
10
Elution in Column Chromatography
11
Distribution of solutes between phases

• Kc distribution constant (partition coefficient
  )
• Define as the molar concentration of analyte in
  the stationary phase divided by the molar
  concentration of the analyte in the mobile phase.

12
Retention Time(????)

• The time taken for the mobile phase to pass
  through the column is called tM (dead time)
• The time between sample injection and an analyte
  peak reaching a detector at the end of the column
  is termed the retention time (tR ).

13
Relating Migration Rates to Distribution Constants
14
Relating Migration Rates to Distribution Constants
15
Retention Factor

• An important factor is widely used to compare the
  migration rates of solutes on columns.
  kA(tR-tM)/tM
• kAlt1 ? elution is so fast that accurate
  determination of the retention time is very
  difficult.
• High retention factors, kAgt20 ? elution
• takes a very long time.
• Ideally, 1ltkAlt5

16
Tailing and Fronting Peaks

• Tailing is caused by sites on the packing that
  have a stronger-than normal retention for the
  solute.
• Fronting is related to the shape of the sorption
  isotherm or that sample introduced onto a column
  is too large.

17
Column Efficiency

• Two related terms, (1) plate height H and (2)
  number of theoretical plates N, are widely used
  to measure the column efficiency.
• NL/H
• Hs2/L

18
Theoretical Plate Model of Chromatography

• Suppose that the chromatographic column contains
  a large number of separate layers, called
  theoretical plates.
• It is important to remember that the plates do
  not really exist.
• Measure column efficiency

19
Calculate Theoretical Plate Numbers

• W width of the peak at its base
• W1/2 peak width at half-height

20
Height Equivalent to Theoretical Plate (HETP)

• Van Deemter equation for plate height
• HETP A B / u C u
• uaverage velocity of the mobile phase
• A, B, and C are factors which contribute to band
  broadening.

21
A - Eddy diffusion

• Molecules move through different paths
• Larger difference in pathlengths for larger
  particles
• This will cause broadening of the solute band,
  because different paths are of different lengths.

22
B - Longitudinal diffusion

• The concentration of analyte is less at the edges
  of the band than at the center.
• Analyte diffuses out from the center to the
  edges.
• Inversely proportional to flow rate - high flow,
  less time for diffusion
• High velocity decreases the effects of
  longitudinal diffusion.

23
C - Resistance to mass transfer

• The analyte takes a certain amount of time to
  equilibrate between the stationary and mobile
  phase.
• The high velocity of the mobile phase lead the
  analyte to have a strong affinity for the
  stationary phase.
• The analyte in the mobile phase will move ahead
  of the analyte in the stationary phase.
• The higher the velocity of mobile phase, the
  worse the broadening becomes.

24
Van Deemter Plots
25
Column Resolution

• Resolution ability to separate two analytes
• High resolution ? Z?W?

26
Optimization of Column Efficiency Resolution
27
Optimization of Column Efficiency Resolution
28
Resolution and Zone Broadening Factors

• u- low flow favors increased resolution
• H (plate height) -use smaller particles,
• lengthen column
• a - vary temperature, composition of
• column/mobile phase
• kA' (capacity factor) - vary temperature,
  composition of column/mobile phase